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Full text of "Laboratory and field studies with 32P labeled Toxorhynchites rutilus rutilus."

474 



Journal of the American Mosquito Control Association Vol. 2, No. 4 



LABORATORY AND FIELD STUDIES WITH 3 2p LABELED 
TOXORHYNCHITES RUTILUS RUTILUS 



B.J. SMITTLE AND D. A. FOCKS 

Insects Affecting Man and Animals Research Laboratory, Agricultural Research Service, U.S. Department 
of Agriculture, P.O. Box 14565, Gainesville, FL 32604 

ABSTRACT. Females and eggs of Toxorhynchites r. rutilus were labeled with 32 P by feeding fourth-stage 
larvae 32 P labeled Aedes aegypti larvae. Eggs from females up to 3 weeks in age had detectable levels of 
radioactivity and individual eggs contained ca. 0.3% of the mother's total radioactivity. Comparisons of 
labeled and unlabeled females in indoor and outdoor cage tests indicated that survival and fecundity of the 2 
groups were approximately equal. No differences were noted for dispersal and fecundity of labeled and 
control females released in field tests. The 32 P-labeled Tx. r. rutilus females behave similarly to unlabeled 
females, and this method of radiolabeling provides a sound tool for tracking laboratory-reared females 
released into an area with an indigenous population. 



INTRODUCTION 

In recent years there has been considerable 
interest in using species of the genus 
Toxorhynchites as predators in the biological 
control of species of mosquitoes that breed in 
artificial and natural containers. Focks et al. 
(1982) obtained ca. 74% control of Aedes aegypti 
(Linn.) and Culex quinquefasciatus Say by the 
addition of one or 2 Toxorhynchites rutilus rutilus 
(Coquillett) larvae to containers in a substan- 
dard housing area. Bailey et al. (1983) ob- 
served that a dense natural population of Tx. r. 
rutilus significantly reduced a natural popula- 
tion of Ae. aegypti in a tire dump when 
compared with 2 other tire dumps with low 
levels of Tx. r. rutilus. 

Ovitraps have been used extensively to 
sample natural populations of Tx. r. rutilus, but 
the results of releasing small numbers of 
laboratory-reared females are often difficult to 
evaluate because of the presence of indigenous 
populations. Since Smittle and Seawright (1983) 
were successful in labeling Ae. aegypti eggs with 
radioactive phosphorus ( 32 P), the use of this 
technique was evaluated to assess its usefulness 
for the identification of eggs from released Tx. 
r. rutilus females. This paper presents the 
results of laboratory labeling of immature Tx. r. 
rutilus with 32 P to produce females that would 
deposit radioactive eggs and the field release of 
radiolabeled females with subsequent recovery 
of radiolabeled eggs. 



METHODS AND MATERIALS 

The Tx. r. rutilus used in these studies were 
reared in the laboratory on a diet of Ae. aegypti 
by methods previously described by Focks et al. 
(1979). Briefly, the Tx. r. rutilus larvae were 
reared on a diet of Ae. aegypti larvae in 50 x 40 
X 10 cm trays of well water at 27.2°C; the 
photoperiod was 14 hr light: 10 hr dark. On 
day 0, 0.05 ml of to 24-hr-old Tx. r. rutilus 



eggs (ca. 330) were added to a rearing tray 
containing 0.15 ml of Ae. aegypti eggs and a 
slurry of 3 g of hydrolyzed yeast and liver 
powder (1:1). Other trays containing only Ae, 
aegypti eggs and food were set on day 0, 3, 5 
and 7. These Ae. aegypti larvae were placed in 
the Tx. r. rutilus trays as required for adequate 
feeding. 

In the preliminary tests, late third- and early 
fourth-stage Ae. aegypti larvae were placed in 
tap water containing either 0.0325 (low) or 
0.0650 (high) |xCi/ml of ™?. After 3 hr the 
larvae were fed a slurry of yeast and liver 
powder. After 48 hr the larvae were removed 
from the 32 P treatment solution, rinsed to 
remove most external radioactivity and offered 
to late third- and early fourth-stage Tx. r. rutilus 
larvae. The Tx. r. rutilus larvae were fed the 
radioactive Ae. aegypti larvae until pupation. 
For emergence, the Tx. r. rutilus pupae were 
placed in water-filled cups in 0.5 x 0.5 x 0.5 
m, clear acrylic cages which were maintained at 
80% RH, 27°C, and with the same photoperiod 
used in larval rearing. The adults were 
provided with cotton rolls soaked with a 50% 
honey-water solution and a black 0.5-liter 
oviposition jar half filled with water. 
Toxorhynchites r. rutilus begin ovipositing 5 or 6 
days after emergence. 

The radioactivity of the various stages was 
measured using a liquid scintillation counter 
having an efficiency for 32 P of greater than 
95%, Only those eggs with counts greater than 
10 counts/min above background were consid- 
ered labeled. The effects of the 32 P-labeling 
were monitored by comparing the daily sur- 
vival and oviposition of the labeled and control 
adults. The radioactivity of the females and 
their eggs during the 2-week period following 
the first oviposition was recorded. 

For each of the 3 field releases conducted, 
similar rearing methods were used but only the 
0.0325 (iCi/ml of 32 P treatment was used for 
radiolabeling the Ae. aegypti larvae. All labeled 



December, 1986 



ToXORHYNCHlTES RUTILUS RUTILUS 



475 



Tx. r. rutilus pupae in the second and third re- 
leases were individually assayed in a G-M or 
proportional counter to assure that adequate 
levels of 32 P would be present in all females. For 
the first 2 releases, pupae were placed in cups 
within 0.5 x 0.5 x 0.5 m screened cages and, 
after emergence, the adults were held in the 
laboratory for about 1 week before release to 
allow for mating and start of oviposition. For 
the third release, the adults were held in screen 
cylinders 48 cm in diameter and 58 cm high in 
an outdoor screened building under ambient 
conditions. 

The approximately square experimental re- 
lease area covering about 12.6 ha on the 
University of Florida campus, has been de- 
scribed previously (Focks et al. 1979). Releases 
were made from the center of a student 
housing complex that occupied an area of 5.3 
ha. This inner area was separated from the 
outer area by roadways. The plot was inter- 
spersed with various hardwood and pine trees, 
shrubs, and open expanses of lawn. It was 
bordered on 3 sides by a more densely wooded 
area and on the fourth side by a lake. 

Oviposition by Tx. r. rutilus was monitored 
with 64 oviposition traps (0.5 liter black jars 
half filled with water) placed on tree boles (ca. 
2 m above the ground) with a uniform spacing 
of ca. 60 m intervals throughout the study site. 
Thirty ovitraps were within the housing area 
(inner area), and the rest were in the outer, 
surrounding wooded areas. The jars were 
checked daily for Tx. r. rutilus eggs, which were 
removed and returned to the laboratory for 
assay of radioactivity. Prior to the releases, the 
ovitraps were monitored for 3 to 5 days to 
detect the presence of indigenous Tx. r. rutilus. 

The Tx. r. rutilus adults were released at sun- 
down in the center of the inner area. Both 32 P 
and control females were released to allow com- 
parison of oviposition and dispersal following 
field release. The number of females released 
was 102 32 P and 102 control, 175 32 P and 67 
control, and 159 32 P and 157 control in the three 
releases, respectively. For each release, 32 P and 
control groups were maintained at the labora- 
tory for oviposition and survival data. 

RESULTS AND DISCUSSION 

Preliminary tests. The mean radioactivity 
levels for the Ae. aegypti larvae and the various 
stages of Tx. r. rutilus are presented in Table 1. 
These data indicate that it is possible to use this 
technique for obtaining uniformly labeled Tx. 
r. rutilus adults. Except for the pupae of Tx. r. 
rutilus, all of the stages from the tray treated 
with 0.0325 fiCi/ml were about one-half as 
radioactive as the stages from the 0.065 u,Ci/ml 



Table 1. Mean radioactivity 3 of Aedes aegypti i larvae 

and various stages of Toxorhynchites r. rutilus 

labeled with 32 P. 



Radioactivity of specimens exposed to: 



Species 

: Stage : 0.0325 n-Ci/ml : 0.065 |xCi/ml 



Ae. aegypti 
IV-stage 

larvae 33,166 ± 3,996 77,845 ± 5,848 
Tx. r. rutilus 
IV-stage 

larvae 114,033 ± 24,842 239,411 ± 33,328 
pupae 25,116 ± 2,027 70,433 ± 14,450 

newly- 
emerged 

adults 22,549 ± 1,323 53,659 ± 4,918 
eggs 
day 1 32.7 ± 2.8 85.1 ± 5.2 

day 13 18.4 ± 3.4 41.9 ± 3.2 

a Counts/min less background and ± standard 
errors. 

treatment. The eggs laid 13 days after oviposition 
commenced had 56% (low dose) and 49% (high 
dose) of the radioactivity present in eggs laid 
on the first day of oviposition. Radioactive 
decay alone would result in eggs retaining 56% 
of the amount present on the first day of 
oviposition, thus substantial amounts of 32 P 
were not lost by the female through routes 
other than oviposition. Eggs from the 0.0325 
p.Ci/ml treatment could be identified for at 
least 2 wk after oviposition began. 

Pupal production and mean daily survival 
for caged adults did not indicate a treatment- 
related reduction due to the 32 P labeling. Total 
oviposition by the radiolabeled females was 
reduced over 14 days by 35 and 61% for the 
low and high doses, respectively, but the 
observed reductions were not significandy 
different from the control (ANOV, P = 0.05) 
due to high day-to-day variability. For the field 
release studies, the lower dose of 0.0325 
u/Ci/ml was used, because it provided adequate 
levels of radioactivity for identification of eggs. 

Field Releases. A summary of the radioac- 
tivity of various stages from the 3 field releases 
is presented in Table 2. The Ae. aegypti larvae 
fed to the Tx. r. rutilus in the first and third 
releases had similar amounts of radioactivity 
and were lower than the amount in larvae used 
for the second release. However, the level of 
radioactivity in Tx. r. rutilus females varied 
considerably, with a range of approximately 
7,000-40,000 counts/minute. Apparendy, slight 
differences in the physiological age of the 
larvae of Tx. r. rutilus can have a marked effect 
on the amount of 32 P label incorporated into 
the adult females and their eggs. Age is an 
important consideration, because consumption 



476 



Journal of the American Mosquito Control Association Vol. 2, No. 4 



Table 


2. Mean 


radioactivity* 


of Aedes aegypti larvae and adult females and 
labeled with 32 P from the 3 field releases. 


eggs of Toxorhynchites 


r. rutilus 




: Stage 




Radioactivity of specimens from indicated release. 




Species 




1 2 




3 



Ae. aegypti 

IV-stage larvae 
Tx. r. rutilus 

adult females 

eggs 



9,333 ± 1,327 

6,923 ± 709 
21.4 ± 1.2 



16,236 ± 2,922 

17,420 ± 969 
60.9 ± 2.2 



9,069 ± 946 

39,494 ± 1,504 
97 ± 6.7 



a Counts/min less background ± standard errors. 

of the radiolabeled prey depends on the 
number of days in fourth stage. Radioactivity 
taken in initially may be eliminated through 
metabolism prior to pupation, and radioactivity 
taken in late may not be absorbed but merely 
passed out of the gut prior to pupation. 

On the first day of oviposition, the amount 
of radioactivity in an individual egg averaged 
about 0.30 ± 0.05% of the radioactivity of the 
female on the first day of oviposition. How- 
ever, 10% of the eggs from females that 
averaged 7,000 counts/min had less than 10 
counts/min above background on the first day 
of oviposition. Therefore, for Field releases, it 
is advisable to use females which have an 
average radioactivity of above 10,000 
disintegrations/min on the first day of oviposition. 

Figure 1 shows the average survival and 
fecundity of caged females from the three 



groups { pooled) that were released in the field. 
Daily fecundity was calculated as the ratio of 
the number of eggs laid on any particular day 
to the number of females alive on that day. 
The daily survivals for labeled and control 
females were virtually identical with the control 
females having slightly higher survival for the 
first 5 days. With the exception of 2 days out of 
14, the 32 P females, had higher fecundity. In 
fact, females from the third release which were 
the highest in radioactivity also had the highest 
survival and oviposition rates. This is in 
contrast with the preliminary studies and 
indicates that differences in fecundity may be 
due to factors other than radiolabeling 
procedures. 

Oviposition in the 64 ovitraps in the release 
study area for the 3 releases ( pooled) is shown 
in Figure 2. Prerelease oviposition by indige- 



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DAYS SINCE RELEASE 

Fig. 1. Survival and fecundity of caged S2 P and control females (average of 3 replicates). 



14 



December, 1986 



TOXORHYNCHITES RUTILUS RUTILUS 



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DAYS SINCE RELEASE 

Fig. 2. Oviposition of 32 P and control females released in housing area (average of 3 releases). 



nous females ranged from 2.3 to 4.0 eggs/day; 
the average daily indigenous oviposition was 
subtracted from the nonradioactive eggs col- 
lected each day to achieve the average eggs/ 
control female released. The control females 
had a higher average oviposition rate initially 
with little difference between the 2 groups on 
the other days. Overall, 1.0 and 0.9 eggs/ 
female released were recovered from the 
ovitrap monitoring system for the control and 
32 P females, respectively. Since cage tests 
indicated that at least 10% of the eggs from 
radioactive females in the first release were 
classified as nonradioactive, the average fecun- 
dity of the 32 P group would have been slightly 
higher and thus there would be little difference 
in fecundity of field released S2 P and control 
females. 

Radioactive eggs were first found in the most 
distant ovitraps 3 to 6 days after release 
indicating movement away from the release 
location at a rate comparable to that of 
unlabeled Tx. r. rutilus observed previously by 
Focks et al. (1979). Some of the 32 P females 
stayed near the release site as radioactive eggs 
were collected nearby throughout the 14-day 
collection period. 

The methods and results presented here 
indicate that it is possible to radiolabel Tx. r. 



rutilus females in the laboratory. These tests 
indicate that 32 P labeled Tx. r. rutilus females, 
ranging between 10,000 and 40,000 disinte- 
grations/min, had survival, dispersal and 
oviposition rates that were comparable to 
unlabeled females. Since the 32 P label provides 
positive identification of laboratory-reared ma- 
terial, the use of 32 P labeled females should be 
a useful tool in field studies with this and 
perhaps other species of Toxorhynchites. 

References Cited 

Bailey, D. L., R. G.Jones and P. R. Simmonds. 1983. 
Effects of indigenous Toxorhynchites rutilus rutilus 
on Aedes aegypti breeding in tire dumps. Mosq. 
News 43:33-37. 

Focks, D. A., S. R. Sackett and D. L. Bailey. 1982. 
Field experiments on the control of Aedes aegypti 
and Culex quinquefasciatus by Toxorhynchites rutilus 
rutilus (Diptera:Culicidae). J. Med. Entomol. 
19:336-339. 

Focks, D. A., J. A. Seawright and D. W. Hall. 1979. 
Field survival, migration and ovipositional charac- 
teristics of laboratory-reared Toxorhynchites rutilus 
rutilus (Diptera: Gulicidae). J. Med. Entomol. 
16:121-127. 

Smittle, B. J. and J. A. Seawright. 1983. Transfer of 
radioactivity to individual eggs by female Aedes 
aegypti treated as larvae with 32 P. Mosq. News 43: 
329-331.